This R21 grant proposal aims to characterize the molecular mechanisms that transform initial alcohol exposures into long-lasting changes in the brain. Our work focuses on a family of chromatin modification enzymes, the histone demethylases (HDM). Epigenetic changes are increasingly being recognized as relevant in addiction and modulation of histone methylation at specific genomic loci often governs changes in gene transcription in response to environmental cues. Because of its ease of genetic manipulation, Drosophila represents a powerful platform on which to study how alterations in chromatin programming result in alcohol- induced changes in behavior. We seek to test the hypothesis that specific HDMs regulate the experience- dependent behavioral changes that accompany exposure to ethanol. To understand the in vivo role of these chromatin modification enzymes in alcohol tolerance and consumption preference, we have begun to systematically knock out every one of the 14 Drosophila HDM genes, all of which have closely related mammalian orthologs. To accomplish this goal, we are employing cutting-edge molecular techniques such a recombineering and homologous recombination. In this exploratory/developmental research grant we propose the following two aims: Aim1 seeks to systematically determine the ethanol tolerance and consumption preference phenotypes of Drosophila strains carrying genetic modifications in all 14 HDM genes. We will accomplish this aim by testing knock-out and overexpression lines for each HDM gene, and by quantitatively assessing their ethanol tolerance and consumption preference. In our preference assay, naive flies show a tendency to avoid food containing 15% ethanol, while ethanol pre-exposure transforms this avoidance into a significant preference. Our preliminary data suggests that at least two HDM knock-out lines show altered ethanol consumption preference. In Aim2, we propose to determine where functionally relevant HDMs bind throughout the genome, by performing chromatin immunoprecipitation coupled with massive parallel deep sequencing (ChIP-seq) using HA-tagged HDM proteins we have in hand. This work will yield a comprehensive map of genes differentially bound by functionally relevant HDMs upon a behavior-changing ethanol exposure. Determining the in vivo functions of HDMs in experience-dependent ethanol plasticity has the potential for high impact on understanding the development of human addiction. These proteins are highly conserved and represent a class of druggable enzymes that hold promise for the future development of therapeutic intervention.